Ion channel clustering at nodes of Ranvier is an essential feature of myelinated axons. Nodal Na+ channel clusters confer several important functional advantages including decreased energy and space requirements, and increased action potential conduction velocity. We recently showed that multiple, overlapping mechanisms contribute to CNS node of Ranvier formation. These mechanisms include both intrinsic (neuronal) and extrinsic (glial mechanisms). Extrinsic mechanisms include interactions between axonal cell adhesion molecules and glia-derived extracellular matrix molecules, as well as an axoglial junction-dependent diffusion barrier that restricts membrane proteins to nodes. Intrinsic mechanisms depend on cytoskeletal and scaffolding proteins. The redundancy of nodal Na+ channel clustering mechanisms makes genetic mouse models the only tractable approach to discover how CNS nodes are formed. Despite these advances, the molecular details for how these mechanisms work remain poorly understood and are even controversial. Here, we will use newly developed genetic mouse models to elucidate the molecular mechanisms responsible for node of Ranvier formation and maintenance. We propose to determine the roles of NF186 and the nodal ECM in node of Ranvier assembly and maintenance. We propose to determine how spectrins function to link the nodal Na+ channel protein complex to the underlying actin cytoskeleton. And finally, we propose to determine how paranodes function as lateral diffusion barriers. The experiments proposed here continue and extend our previous studies that identified multiple, overlapping mechanisms for CNS node of Ranvier formation.